Wu D Y, Wallace R B
Department of Molecular Biochemistry, Beckman Research Institute of the City of Hope, Duarte, California 91010.
Genomics. 1989 May;4(4):560-9. doi: 10.1016/0888-7543(89)90280-2.
A novel DNA sequence detection method that utilizes the ligation of oligonucleotide pairs that are complementary to adjacent sites on appropriate DNA templates is described. The product is increased by either linear or exponential amplification using sequential rounds of template-dependent ligation. In the case of linear amplification, a single pair of oligonucleotides is ligated, the reaction is heated to dissociate the ligation product, and an additional round of ligation is performed. After n rounds there is a (1 + x) X n-fold amplification of product, where x is the efficiency of the ligation reaction. Exponential amplification utilizes two pairs of oligonucleotides, one complementary to the upper strand and one to the lower strand of a target sequence. The products of the ligation reaction serve as templates for subsequent rounds of ligation. In this case there is (1 + x)(n-1)-fold amplification of product after n rounds. A single base-pair mismatch between the annealed oligonucleotides and the template prevents ligation, thus allowing the distinction of single base-pair differences between DNA templates. At high template concentrations, the ligation reaction has an efficiency approaching 100%. In this report, we demonstrate the use of the ligation amplification reaction (LAR) to distinguish the normal from the sickle cell allele of the human beta-globin gene. We also report the use of LAR as a detection system for polymerase chain reaction-enriched DNA sequences.
本文描述了一种新颖的DNA序列检测方法，该方法利用与合适DNA模板上相邻位点互补的寡核苷酸对的连接。通过连续进行依赖模板的连接轮次，产物可通过线性或指数扩增得以增加。在进行线性扩增时，一对寡核苷酸进行连接，反应加热以解离连接产物，然后进行新一轮的连接。经过n轮后，产物会有(1 + x)×n倍的扩增，其中x是连接反应的效率。指数扩增利用两对寡核苷酸，一对与靶序列的上链互补，另一对与下链互补。连接反应的产物作为后续连接轮次的模板。在这种情况下，经过n轮后产物会有(1 + x)(n - 1)倍的扩增。退火的寡核苷酸与模板之间单个碱基对的错配会阻止连接，从而能够区分DNA模板之间单个碱基对的差异。在高模板浓度下，连接反应的效率接近100%。在本报告中，我们展示了使用连接扩增反应（LAR）来区分人类β-珠蛋白基因的正常等位基因和镰状细胞等位基因。我们还报告了将LAR用作聚合酶链反应富集的DNA序列检测系统的情况。
